WO2016187499A1 - Régulation glycémique améliorée à l'aide d'un facteur intrinsèque lié à un conjugué de vitamine b12 d'un agoniste du récepteur du glucagon-like peptide-1 - Google Patents

Régulation glycémique améliorée à l'aide d'un facteur intrinsèque lié à un conjugué de vitamine b12 d'un agoniste du récepteur du glucagon-like peptide-1 Download PDF

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WO2016187499A1
WO2016187499A1 PCT/US2016/033426 US2016033426W WO2016187499A1 WO 2016187499 A1 WO2016187499 A1 WO 2016187499A1 US 2016033426 W US2016033426 W US 2016033426W WO 2016187499 A1 WO2016187499 A1 WO 2016187499A1
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peptide
conjugate
pharmaceutical composition
conjugated
analog
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PCT/US2016/033426
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English (en)
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Robert Doyle
Jonathan D. Bortz
David S. Hermelin
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Syracuse University
Xeragenx Llc
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Priority to US15/575,468 priority Critical patent/US20180154007A1/en
Publication of WO2016187499A1 publication Critical patent/WO2016187499A1/fr
Priority to US17/239,934 priority patent/US11771772B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention encompasses compositions and methods for lowering blood glucose. More specifically, the present invention provides compositions comprising a complex comprising IF and B12 conjugated to a peptide comprising a GLP-1 receptor agonist.
  • the composition may be delivered subcutaneously.
  • Diabetes mellitus is a chronic metabolic disorder caused by an absolute or relative deficiency of insulin hormone. Insulin is produced by the beta cells of the islets of Langerhans located in the pancreas, and the absence, destruction, or other loss of these cells results in type 1 DM, or insulin-dependent diabetes mellitus (IDDM). Most children with diabetes have IDDM and a lifetime dependence on regular injections of exogenous insulin. Overall incidence is at least 15 cases per 100,000 individuals annually and probably increasing. An estimated 3 children out of 1000 develop IDDM by age 20 years. More than 700,000 Americans have type 1 DM.
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 DM or non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder. Most patients with NIDDM have insulin resistance, and their beta cells lack the ability to overcome this resistance. Although this form of diabetes was previously uncommon in children, in some, countries 20% or more of new patients with diabetes in childhood and adolescence have NIDDM, a change associated with increased rates of obesity. Other patients may have inherited disorders of insulin release leading to maturity onset diabetes of the young (MODY). Of the total incidence of diabetes in the U.S., NIDDM accounts for about 90%, whereas IDDM accounts for the remaining 10%.
  • GLP-1 Glucagon-like peptide-1
  • GLP-1 (7-36)amide also inhibits gastric acid secretion and gastric emptying, as well as suppressing glucagon release (therefore reducing hepatic-derived glucose) and promoting an increase in pancreatic ⁇ -cell mass.
  • GLP-1 (7-36)amide
  • glucagon release therefore reducing hepatic-derived glucose
  • pancreatic ⁇ -cell mass As a therapeutic target, there has been substantial interest in GLP-1 , since actions of GLP-1 (inhibition of gastric emptying; decreased blood glucose concentration) are preserved in subjects with poorly controlled Type 2 Diabetes.
  • agonists for the GLP-1 receptor are available, there is a need in the art for GLP-1 receptor agonists with improved efficacy and pharmacokinetics.
  • FIG. 1 depicts the structure of Vitamin Bi 2 showing the Co (III) atom coordinated by the four nitrogens of the corrin ring, dimethylbenzimidazole (DMB), and the X group which can be a cyanide, methyl, or deoxyadenosyl groups and a schematic of the synthesis of Bi 2 -Ex-4 conjugates with the following reagents and conditions: (i) EDCI, HOBt, propargyl amine, rt, DMSO, 16 h; (ii) EDCI, HOBt, 1 - amino-3-butyne, rt, DMSO, 16 h; (iii) EDCI, HOBt, 1 -amino-4-pentyne, rt, DMSO, 16 h; (iv) EDCI, HOBt, 1 -amino-5-hexyne, rt, DMSO, 16 h; (v) (2), CuS0 4 ,
  • FIG. 2 depicts an LC trace showing purified compound 7 as a monomer (7.5 min) and dimer (6.5 min) and ESI MS (inset) of compound 7 showing m/z of 1415.5 Da, which corresponds to the +4 of compound 7 yielding a consistent mass of 5658 Da;
  • FIG. 3A depicts a graph of the EC 50 curves for compounds 1 , 6, 7, 8, and 9.
  • FIG. 3B depicts a plot of the EC50 values for compounds 1 , 6, 7, 8, and 9 (27, 121 , 68, 246 and 271 pM, respectively).
  • FIG. 4A depicts a graph of the binding affinities of the B 2 binding protein, IF, to compound 7 and to B 2 (as cyanocobalamin), namely 6.8 and 0.12 nM, respectively.
  • FIG. 4B depicts a graph of the EC50 values for compound 7, IF-7 and compound 1 , which were 68, 125, and 27 pM, respectively.
  • the present invention comprises the use of intrinsic factor to enhance the efficacy of a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist following administration.
  • a peptide of interest is conjugated to Bi 2 (or a B-I2 analog) and the B 2 conjugate is then complexed with intrinsic factor.
  • Such a composition is suitable for subcutaneous administration and shows enhanced activity at lowering blood glucose relative to a composition that doesn't comprise intrinsic factor.
  • the invention provides a method of lowering blood glucose following administration of a pharmaceutical composition comprising a complex comprising IF and a B 2 conjugate, wherein the B 2 is conjugated to a peptide comprising a GLP-1 receptor agonist.
  • the pharmaceutical compositions disclosed herein enable a rapid and significant drop in glucose levels following administration.
  • the present invention encompasses a pharmaceutical composition comprising intrinsic factor and Bi 2 or an analog thereof, wherein the Bi 2 or analog thereof is conjugated to a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist.
  • B 2 analogs may be modified to improve conjugation chemistry, bioavailability, solubility, stability, handling properties, or a combination thereof, as compared to an unmodified version of Bi 2 .
  • a composition of the invention comprises a modified B 2 or B 2 analog.
  • a composition of the invention comprises a prodrug of B 2 or a B 2 analog.
  • composition of the invention may further comprise a
  • composition of the invention may contain preserving agents, solubilizing agents, stabilizing agents, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, or antioxidants.
  • vitamin B 12 cobalamin
  • Vitamin B 2 is a water-soluble vitamin with a highly complex structure, comprising a midplanar corrin ring composed of four pyrroline elements linked to a central cobalt(lll) atom.
  • vitamin Bi 2 , B12 and cobalamin may be used interchangeably.
  • the central cobalt(lll) atom is six- coordinated, with the equatorial positions filled by the nitrogen atoms of the corrin macrocycle.
  • the (conventionally) 'lower', 'a'-axial site is occupied by an imidazole nitrogen atom from a 5',6'-dimethylbenzimidazole (DMB) base whereas the 'upper', ' ⁇ '-axial site can be occupied by various X groups (e.g. CN “ , CH 3 " , Ado " , SCN “ , SeCN “ , SO3 “ and thiourea).
  • the corrin ring incorporates seven amide side chains, three acetamides (a, c, g) and four propionamides (b, d, e, f).
  • the four pyrrole rings are usually indicated as A, B, C and D, as shown in FIG. 1 .
  • a modification of Bi 2 may be any modification on B 2 that improves conjugation chemistry, bioavailability, solubility, stability, handling properties, or a combination thereof.
  • conjugation of Bi 2 describes the conjugation of a peptide to Bi 2 or modified Bi 2 .
  • a Bi 2 conjugate of the invention may be modified and/or conjugated at a propionamide, acetamide, hydroxyl group, the cobalt(lll) ion and the phosphate moiety, provided the B 2 conjugate binds IF.
  • modification or conjugation sites for a B 2 conjugate of the invention include at the a-position or b- position on the A-ring, at the c-position or cf-position on the B-ring, at the e-position on the C-ring, at the g-position on the D ring, at the -position, at the phosphate moiety, at the 5'- or 2'-hydroxyl on the ribose, and at the cobalt ion.
  • Preferred sites of modification and/or conjugation may include sites on the A ring such as the b- position, sites on the C ring such as the e-position, sites on the ribose unit such as the 5'-hydroxyl group, and the cobalt cation.
  • the e-position may be modified and/or conjugated to allow interaction with IF.
  • other sites of modification and/or conjugation may be utilized provided they maintain the binding affinity of Bi 2 for IF.
  • the 5'-hydroxyl on the ribose may be modified to a carboxylic acid. The resulting carboxylic acid may then be used to conjugate B 2 to a peptide. A method of performing this is described in more detail below and in the Examples.
  • cyanocobalamin (CN)Cbl)
  • anhydrides furnishing unstable ethers.
  • Another method for conjugation may be the carbamate or carbonate methodology as described by Russell-Jones (WO
  • the hydroxyl group at position 5' is first reacted with a carbonyl group equivalent - 1 , 1 '- carbonyldiimidazole (CDI) or 1 , 1 '-carbonylbis(1 ,2,4-triazole) (CDT) - and then treated with an amine or an alcohol giving carbamates and carbonates, respectively, at the 5'-position of the ribose tail.
  • CDI carbonyl group equivalent - 1 , 1 '- carbonyldiimidazole
  • CDT 1 , 1 '-carbonylbis(1 ,2,4-triazole)
  • the 5'-OH group can be oxidized to the corresponding carboxylic acid using the 2-iodoxybenzoic acid (IBX) / 2- hydroxypyridine (HYP) system as an oxidant and then coupled with amines.
  • IBX 2-iodoxybenzoic acid
  • HEP 2- hydroxypyridine
  • B-I2 or an analog thereof and a peptide may be: i) conjugated directly together; or ii) held apart by a 'linker' to produce distance between the B 2 or an analog thereof and the peptide.
  • the peptide is described in more detail in
  • Bi 2 or an analog thereof may be conjugated to a peptide directly.
  • an intervening amino acid sequence or linker can be used to conjugate the peptide to B 2 . It is to be understood that conjugation of the Bi 2 or an analog thereof to a peptide will not adversely affect either the binding function of the Bi 2 or an analog thereof to IF or the function of the peptide.
  • Suitable linkers include, but are not limited to, amino acid chains and alkyl chains functionalized with reactive groups for conjugating to both the B 2 or analog thereof and the peptide.
  • the linker may include amino acid side chains, referred to as a peptide linker. Accordingly, amino acid residues may be added to B 2 or an analog thereof for the purpose of providing a linker by which B 2 or an analog thereof can be conveniently affixed to a peptide.
  • Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
  • the linker may be an alkyl chain linking group.
  • a first functional group on the alkyl chain such as a carboxyl group or an activated ester.
  • a peptide may be attached to the alkyl chain to complete the formation of a conjugate by reacting a second functional group on the alkyl chain with an appropriate group on the peptide.
  • the second functional group on the alkyl chain is selected from substituents that are reactive with a functional group on the peptide while not being reactive with B 2 or an analog thereof.
  • the second functional group of the alkyl chain linking group can be an amino group.
  • a carboxylic acid group generated from the oxidation of the 5'-hydroxyl of the ribose on B 2 with a bifunctional amine-alkyne of variable methylene spacer length, a B 2 -alkyne compound may be generated.
  • a peptide may be attached to the alkyl chain to complete the formation of the conjugate by reaction a second functional group on the alkyl chain with an
  • the second functional group on the alkyl chain is an alkyne.
  • a peptide modified with a functional group that reacts with an alkyne may then be conjugated to the alkyl chain.
  • a peptide modified with an azido group may be conjugated to the alkyne group on the alkyl chain.
  • linking groups may alternatively be coupled first to the peptide and then to B12 or an analog thereof.
  • An alkyl chain linking group may be one to 40 or more atoms long, more often 1 to 10 atoms long.
  • an alkyl chain linking group may comprise one or more heteroatoms (e.g. O, N, S).
  • an alkyl chain linking group may be 1 , 2, 3, 4, 5, 6 or 7 atoms long.
  • an alkyl chain linking group may comprise 3, 4, 5, or 6 carbons.
  • an alkyl chain linking group may comprise 4 carbons.
  • An alternative chemical linking group to an alkyl chain is polyethylene glycol (PEG), which is functionalized in the same manner as the alkyl chain described above for incorporation in the conjugates.
  • PEG polyethylene glycol
  • Bi 2 or an analog thereof may be PEGylated for improved systemic half-life and reduced dosage frequency.
  • PEG may be added to a linker.
  • B 2 or an analog thereof may comprise a linker and PEG.
  • B 2 or an analog thereof may be conjugated to a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist.
  • GLP-1 glucagon-like peptide-1
  • peptide is meant an amino acid sequence that includes 5 or more amino acid residues.
  • a peptide may comprise about 5 or more amino acids.
  • a peptide may comprise about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, or about 100 or more amino acids.
  • a peptide may comprise 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids.
  • a peptide may comprise from about 30 to about 60 amino acids.
  • a peptide may comprise from about 35 to about 55 amino acids.
  • a peptide may comprise from about 40 to about 55 amino acids.
  • a peptide may comprise about 45 amino acids.
  • a peptide comprises 45 amino acids.
  • a peptide may be ordered (a-helix or ⁇ -sheet), disordered, or a combination thereof.
  • a peptide may adopt an a-helical structure.
  • a peptide may adopt an a-helical structure with a disordered terminal end.
  • a peptide may comprise a glucagon-like peptide-1 (GLP-1 ) receptor agonist.
  • GLP-1 receptor agonist may also be referred to as an incretin mimetic.
  • GLP-1 is an incretin derived from the transcription product of the
  • GLP-1 receptor agonist interacts with and is an agonist of the GLP-1 receptor and may be used for the treatment of type 2 diabetes. Any suitable GLP-1 receptor agonist may be used in the invention.
  • GLP- 1 receptor agonists may include exendin-4 (EX-4), exenatide (Byetta/Bydureon), liraglutide (Victoza, Saxenda), lixisenatide (Lyxumia), albiglutide (Tanzeum), dulaglutide (Trulicity), and taspoglutide.
  • a GLP-1 receptor agonist may be an exendin-4 (EX-4) peptide.
  • a GLP-1 receptor agonist may comprise the sequence set forth in SEQ ID NO:2
  • HGEGTFTSDLSKQMEEEAVRLFIENWLKNGGPSSGAPSSGAPPPS HGEGTFTSDLSKQMEEEAVRLFIENWLKNGGPSSGAPSSGAPPPS.
  • a GLP-1 receptor agonist of the invention may be modified to facilitate conjugation to B-I2.
  • a GLP-1 receptor agonist may comprise the sequence set forth in SEQ ID NO:1
  • HGEGTFTSDLS(Kazido)QMEEEAVRLFIENWLKNGGPSSGAPSSGAPPPS wherein the lysine at the 12 position is modified with an azido group.
  • a peptide of the invention may be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
  • the invention encompasses any of a variety of forms of peptide derivatives that include amides, cyclized peptides, polymerized peptides, conservatively substituted variants, analogs, fragments, peptoids, chemically modified peptides, and peptide mimetics.
  • a peptide can be modified by terminal-NH 2 acylation (e.g., acetylation, or thioglycolic acid amidation) or by terminal-carboxylamidation (e.g., with ammonia, methylamine, and the like terminal modifications).
  • Peptides of the invention may comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non- genetically encoded amino acids, and combinations thereof. Peptides may include both L-form and D-form amino acids.
  • Non-genetically encoded amino acids may include but are not limited to 2-aminoadipic acid; 3-aminoadipic acid; ⁇ - aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid); 6- aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo- isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline;
  • Representative derivatized amino acids may include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine can be derivatized to form N-im- benzylhistidine.
  • the term “conservatively substituted variant” refers to a peptide comprising an amino acid residue sequence similar to a sequence of a reference peptide in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the same or similar targeting activity.
  • the phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide displays targeting activity.
  • Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • Peptides of the present invention may be synthesized by any of the techniques that are known to those skilled in the art of peptide synthesis. Synthetic chemistry techniques, such as a solid-phase Merrifield- type synthesis, may be preferred for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. A summary of representative techniques can be found in Stewart & Young (1969) Solid Phase Peptide Synthesis. Freeman, San Francisco; Merrifield (1969) Adv Enzymol Relat Areas Mol Biol 32:221 -296; Fields & Noble (1990) Int J Pept Protein Res 35:161 - 214; and Bodanszky (1993) Principles of Peptide Synthesis. 2nd rev. ed. Springer- Verlag, Berlin; New York. Solid phase synthesis techniques can be found in
  • IF is bound to a Bi 2 conjugate of the invention thereby forming a complex.
  • a complex of the invention comprises IF complexed to a B 2 conjugate of the invention.
  • Intrinsic factor (IF) is a glycosylated protein that is secreted from the gastric mucosa and the pancreas.
  • IF binds Bi 2 with picomolar affinity (K d ⁇ 1 pM).
  • the IF protein facilitates transport of B 2 across the intestinal enterocyte, which occurs by receptor-mediated endocytosis at the apically expressed IF-B 2 receptor (cubilin).
  • Cubilin works to transport Bi 2 in concert with an anchoring protein amnionless (Am). Following transcytosis, and between 2.5 and 4 h after initial ingestion, Bi 2 appears in blood plasma bound to the third trafficking protein, transcobalamin II (TCII).
  • TCII transcobalamin II
  • the inventors have discovered that conjugating a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist to Bi 2 which is then complexed with IF, enables a rapid and significant drop in glucose levels following subcutaneous administration. This is in contrast to the same peptide that when administered conjugated to B 2 demonstrated no activity following subcutaneous administration.
  • GLP-1 glucagon-like peptide-1
  • the IF may be complexed to Bi 2 before or after conjugation of Bi 2 or an analog thereof to a peptide.
  • IF may be
  • IF may be pre-complexed to a B 2 conjugate by combining the conjugate with IF in solution.
  • Bi 2 conjugate may be combined with IF in PBS at pH 7.4 or in MES buffer at pH 5.5 or in water at pH 8 at temperatures ranging from about 25°C to about 37°C.
  • IF may be contacted with B 2 conjugate for at least 30 minutes.
  • IF may be contacted with Bi 2 conjugate for at least 5 min, at least 10 min, at least 15 minutes, at least 20 min, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours or at least 6 hours.
  • IF and B-12 conjugate may be pre-complexed.
  • IF and the B12 conjugate may be combined in solution.
  • a greater amount of IF may be added to solution relative to Bi 2 conjugate. For example, the ratio of IF to Bi 2
  • conjugate may be 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1,2:1,2.5:1, 3:1,3.5:1,4:1,4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1 , 20:1 , 25:1 , 30:1, 35:1 , 40:1 , 45:1 , 50:1 , 60:1 , 70:1, 80: 1 , 90: 1 , or 100: 1.
  • the ratio of IF to B 2 conjugate may be 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1 , 2.5:1 , 3:1 , 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
  • an excess of 5% or more IF relative to Bi 2 conjugate may be added to solution.
  • an excess of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90% or 100% IF relative to B 12 conjugate may be added to solution.
  • an excess of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% IF relative to B 12 conjugate may be added to solution.
  • excess IF is added to the solution relative to Bi 2 conjugate.
  • the ratio of B 12 conjugate to IF may be 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 , 2:1 , 2.5:1 , 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1 , 25:1 , 30:1 , 35:1 , 40:1 , 45:1 , 50: 1 , 60: 1 , 70: 1 , 80: 1 , 90: 1 , or 100: 1.
  • the ratio of B 12 conjugate to IF may be 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
  • an excess of 5% or more B 2 conjugate relative to IF may be added to solution. For example, an excess of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90% or 100% B 12
  • conjugate relative to IF may be added to a solution.
  • an excess of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% B 12 conjugate relative to IF may be added to a solution.
  • Prior to administration of a composition of the invention it may be necessary to remove un-complexed IF and/or un-complexed B 2 conjugate.
  • IF of the invention may be expressed and purified via standard methodology.
  • IF of the invention is in a pharmaceutically acceptable form.
  • IF is expressed in an environment that is not contaminated with other B12 binding proteins or B12 analogs, is extracted in an environment that is not contaminated with other B-12 binding proteins, and is scalable and reproducible per applicable regulatory standards.
  • IF may be expressed and purified from a transgenic plant, such as
  • IF may be from any species, provided it binds to B 2 and human cubilin.
  • IF can be found in a variety of species. Non-limiting examples include human
  • NP_005133.2 mouse (P52787.2), rat (NP_058858.1 ), dog (Q5XWD5.1 ), cat (XP_003993466.1 ), cattle (NP_001 193168.1 ), non-human primates (EHH56203.1 , XP_004051305.1 ), and horse (XP_0085081 17.1 ).
  • sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit.
  • "percent identity" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl.
  • BLAST nucleotide searches may be performed with the BLASTN program to obtain nucleotide sequences homologous to a nucleic acid molecule of the
  • BLAST protein searches may be performed with the BLASTX program to obtain amino acid sequences that are homologous to a polypeptide of the invention.
  • Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402,
  • a homolog has at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or 89% identity to human IF.
  • a homolog has at least 90%, at least 91 at least%, at least 92 at least%, at least 93 at least%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to human IF.
  • a homolog may have at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or 89% identity to human IF.
  • a homolog has at least 90%, at least 91 at least%, at least 92 at least%, at least 93 at least%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the IF sequence accession number NP_005133.2.
  • the IF comprises the sequence disclosed in accession number NP_005133.2.
  • the IF comprises the sequence disclosed in accession number NP_005133.2 but for one to 10 conservative amino acid substitutions.
  • the IF comprises the sequence disclosed in accession number NP_005133.2 but for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaces with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g.
  • lysine, arginine, histidine acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, histidine).
  • the resulting peptide comprising the substitution should have similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionally of the peptide does not significantly change.
  • a skilled artisan would be able to determine amino acids essential to Bi 2 binding to ensure binding to Bi 2 or a Bi 2 conjugate.
  • the present disclosure also provides pharmaceutical
  • compositions comprising a complex of intrinsic factor and a Bi 2 conjugate, as an active ingredient, and at least one pharmaceutically acceptable excipient.
  • the compositions may be formulated for subcutaneous or intraperitoneal administration.
  • Compositions for subcutaneous or intraperitoneal administration are generally in the form of a liquid.
  • the pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, taste-masking agent, a flavoring agent, or a coloring agent.
  • the amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.
  • the excipient may be a diluent.
  • the diluent may be compressible (i.e., plastically deformable) or abrasively brittle.
  • suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,
  • suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate.
  • the excipient may be a binder.
  • Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, Ci 2 -Ci 8 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.
  • the excipient may be a filler.
  • suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone.
  • the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.
  • the excipient may be a buffering agent.
  • suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).
  • the excipient may be a pH modifier.
  • the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
  • the excipient may be a disintegrant.
  • the disintegrant may be non-effervescent or effervescent.
  • Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth.
  • suitable effervescent disintegrants include sodium
  • the excipient may be a dispersant or dispersing enhancing agent.
  • Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.
  • the excipient may be a preservative.
  • suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.
  • the excipient may be a lubricant.
  • suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.
  • the excipient may be a taste- masking agent.
  • Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers; cellulose acetate phthalate; and combinations thereof.
  • the excipient may be a flavoring agent.
  • Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.
  • the excipient may be a coloring agent.
  • Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).
  • the weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1 % or less of the total weight of the composition.
  • compositions can be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient.
  • Such compositions can be administered parenterally (i.e. subcutaneous and intraperitoneal) in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • parenterally i.e. subcutaneous and intraperitoneal
  • dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18 th ed, 1995), and Liberman, H. A. and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).
  • a composition may be an oral composition.
  • the preparation may be an aqueous or an oil-based solution.
  • Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as
  • etheylenediaminetetraacetic acid etheylenediaminetetraacetic acid
  • a buffer such as acetate, citrate, or phosphate
  • an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol.
  • the pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
  • Oil- based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil.
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • a composition comprising a complex of intrinisic factor and a B 2 conjugate is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition.
  • a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition.
  • suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating compositions into delivery vehicles are known in the art.
  • a liposome delivery vehicle may be utilized.
  • Liposomes depending upon the embodiment, are suitable for delivery of a complex of intrinsic factor and a Bi 2 conjugate in view of their structural and chemical properties.
  • liposomes are spherical vesicles with a phospholipid bilayer membrane.
  • the lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells.
  • a complex of intrinsic factor and a Bi 2 conjugate may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell's membrane.
  • Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths.
  • Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA),
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • DPG diphosphatidylglycerol
  • PC phosphatidylcholine
  • phosphatidylethanolamine PE
  • the fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated.
  • Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate), n- hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9, 12-octadecandienoate
  • the two fatty acid chains of a phospholipid may be identical or different.
  • Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyi PS, distearoyi PC, dimyristoyi PS, dimyristoyi PC, dipalmitoyi PG, stearoyi, oleoyl PS, palmitoyl, linolenyl PS, and the like.
  • the phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids.
  • egg yolk is rich in PC, PG, and PE
  • soy beans contains PC, PE, PI, and PA
  • animal brain or spinal cord is enriched in PS.
  • Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used.
  • phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1 -(2,3- dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride, 1 , 1 '-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchloarate, 3,3'-deheptyloxacarbocyanine iodide, 1 , 1 '-dedodecyl-3,3,3',3'- tetramethylindocarbocyanine perchloarate, 1 , 1 '-dioleyl- 3,3,3',3'-tetramethylindo carbocyanine methanesulfonate, N-4- (delinoleylaminostyryl)-N-methylpyri
  • cationic lipids such as N-(1 -(2,3- dioleolyoxy)
  • Liposomes may optionally comprise sphingolipids, in which spingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes.
  • Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.
  • Liposomes may further comprise a suitable solvent.
  • the solvent may be an organic solvent or an inorganic solvent.
  • Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.
  • Liposomes carrying a complex of intrinsic factor and a Bi 2 conjugate may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241 ,046, 4,394,448, 4,529,561 , 4,755,388, 4,828,837, 4,925,661 , 4,954,345, 4,957,735, 5,043, 164, 5,064,655, 5,077,21 1 and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety.
  • liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing.
  • the liposomes are formed by sonication.
  • the liposomes may be multilamellar, which have many layers like an onion, or unilamellar.
  • the liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar lipsomes.
  • liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.
  • a composition of the invention may be delivered to a cell as a microemulsion.
  • Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and "oil.”
  • the "oil” in this case, is the supercritical fluid phase.
  • the surfactant rests at the oil-water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art.
  • the aqueous microdomains suitable for use in the invention generally will have characteristic structural dimensions from about 5 nm to about 100 nm.
  • microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates.
  • the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil.
  • the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant.
  • the "oil" of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions.
  • a complex of intrinsic factor and a B 2 conjugate may be
  • a complex of intrinisic factor and a B-I2 conjugate may be delivered in a dendritic macromolecule, or a dendrimer.
  • a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe.
  • the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the
  • phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments.
  • Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the invention therein.
  • dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape.
  • the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis.
  • a variety of dendrimer sizes are suitable for use in the invention. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.
  • a composition of the invention may be used in a method to lower blood glucose in a subject.
  • the method comprises administering an effective amount of a pharmaceutical composition comprising intrinsic factor complexed with Bi 2 or an analog thereof, wherein the Bi 2 or analog thereof is conjugated to a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist.
  • the composition may be administered
  • a composition of the invention may be used in a method for treating diabetes meilitus in a subject.
  • the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising intrinsic factor complexed with B 2 or an analog thereof, wherein the B 2 or analog thereof is conjugated to a peptide comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist.
  • the composition may be administered subcutaneously or intraperitoneally.
  • Diabetes meilitus (DM) is a chronic metabolic disorder caused by an absolute or relative deficiency of insulin hormone.
  • Insulin is produced by the beta cells of the islets of Langerhans located in the pancreas, and the absence, destruction, or other loss of these cells results in type 1 DM, or insulin-dependent diabetes meilitus (IDDM).
  • IDDM insulin-dependent diabetes meilitus
  • Type 2 DM, or non-insulin- dependent diabetes meilitus (NIDDM) is a heterogeneous disorder. Most patients with NIDDM have insulin resistance, and their beta cells lack the ability to overcome this resistance.
  • lowering blood glucose is meant that blood glucose is lowered by more than 1 -fold relative to no treatment.
  • blood glucose is lowered by more than 1 -fold, more than 1 .5-fold, more than 2-fold, more than 2.5- fold, more than 3-fold, more than 3.5-fold, more than 4-fold, more than 4.5-fold or more than 5-fold relative to no treatment.
  • blood glucose is lowered by more than 1 -fold, more than 1 .1 -fold, more than 1 .2-fold, more than 1 .3- fold, more than 1 .4-fold, more than 1 .5-fold, more than 1.6 fold, more than 1 .7-fold, more than 1 .8-fold, more than 1 .9-fold or more than 2-fold relative to no treatment.
  • lowering of blood glucose by administering a peptide within a complex is increased relative to a peptide alone or a peptide conjugated to Bi 2 .
  • a peptide within a complex may lower blood glucose by more than 1 -fold relative to a peptide conjugated to Bi 2 .
  • a peptide within a complex may lower blood glucose by more than 1 -fold, more than 1 .5-fold, more than 2-fold, more than 2.5-fold, more than 3-fold, more than 3.5-fold, more than 4-fold, more than 4.5-fold or more than 5-fold relative to a peptide conjugated to Bi 2 .
  • a peptide within a complex may lower blood glucose by more 1 -fold, more than 1 .1 -fold, more than 1.2-fold, more than 1 .3- fold, more than 1 .4-fold, more than 1 .5-fold, more than 1.6 fold, more than 1 .7-fold, more than 1 .8-fold, more than 1 .9-fold or more than 2-fold relative to a peptide conjugated to Bi 2 .
  • the bioavailability of GLP-1 receptor agonists following subcutaneous administration is generally poor.
  • the methods disclosed herein provide a means to subcutaneously deliver a GLP-1 receptor agonist that enables a rapid and significant drop in glucose levels.
  • the complex comprising IF and B 2 conjugated to a peptide comprising a GLP-1 receptor agonist increases the bioavailability of the peptide relative to peptide conjugated to Bi 2 . Accordingly, the bioavailability of the peptide conjugated to B 2 and complexed with IF is increased relative to a peptide
  • Suitable Bi 2 conjugates include Bi 2 -peptide conjugates as described above, and also may include B-i 2 -small molecule conjugates. Importantly, such B-i 2 -small molecule conjugates must (a) maintain biological activity of the small molecule, and (b) maintain binding of the conjugate to IF. Suitable B 2 -small molecule conjugates are known in the art.
  • a complex of the present method may be administered subcutaneously, intraperitoneally, or another parental administration route.
  • a method of the invention may be used lower blood glucose in a subject that is a human, a livestock animal, a companion animal, a lab animal, or a zoological animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoological animal.
  • a "zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears.
  • the animal is a laboratory animal.
  • Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates.
  • the animal is a rodent.
  • Non-limiting examples of rodents may include mice, rats, guinea pigs, etc.
  • the subject is a human.
  • the term "effective amount” means an amount of a peptide that leads to measurable and beneficial effects for the subject administered the substance.
  • the effective amount or dose of peptide administered according to this discovery will be determined by the circumstances surrounding the case, including the peptide administered, the status of the symptoms being treated, the medical history of the subject as well as other considerations known by a skilled artisan.
  • a composition of the disclosure may be administered as multiple doses. Administration may be daily, twice-daily, three times per day, weekly, twice weekly, monthly, twice monthly, or more.
  • the duration of treatment can and will vary depending on the subject and disease to be treated. For example, the duration of treatment may be for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.
  • the duration of treatment may be for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months.
  • the duration of treatment may be for 1 year, 2 years, 3 years, 4 years, 5 years, or greater than 5 years. It is also contemplated that administration may be frequent for a period of time and then administration may be spaced out for a period of time.
  • Compounds 2-5 were purified by RP-HPLC using a C18 column monitoring at 360 nm. A mobile phase of 0.1 % TFA water was used with a flow rate of 1 mL/min and a gradient of 0-13% acetonitrile over 13 minutes. Compounds 2-5 were then characterized by 1 H NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-Tof MS).
  • MALDI-Tof MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • FIG. 4A shows that compound 7 maintained significant IF binding (6.8 nM), albeit diminished compared to native B 2 (as cyanocobalamin; 0.12 nM).
  • agonism of the GLP-1 R was established.
  • IF-7 displayed significant agonism (EC 50 125 pM) of the GLP-1 R. This is indeed the first example confirming pre-binding of a B 2 -peptide conjugate to a B 2 dietary protein (such as IF) maintains in vitro function (in this GLP1 -R agonism) and is highly significant in its potential applications to Bi 2 based drug development in general.
  • the present invention demonstrates that a series of B-
  • Azido modified K12 Ex-4 displays comparable agonism of the GLP-1 R to unmodified Ex-4, which is key to the facile syntheses described.
  • a four conjugate QSAR study indicated that distance between Bi 2 and Ex-4 was an important parameter in maintaining Ex-4 agonism of GLP-1 R.
  • An optimized conjugate was selected for IPGTT screening in vivo and investigated, both as free conjugate and bound to gastric IF protein.
  • Conjugation of B12 to exendin-4 may also modify brain uptake or localization with the effect of reducing Ex-4 activity in the brain. Such a result would likely lead to a loss of the nausea seen with Ex-4, while still allowing
  • Glutamax-I with 4.5 g/L D-glucose, 1 10 mg/L sodium pyruvate, and 0.1 % Geneticin (Life Technologies). About 60,000 cells were plated in each well of a 96 well plate.
  • HEK cells stably transfected with human GLP-1 R were used to test the potency of each new conjugate.
  • the HEK-GLP-1 R cells were grown in DMEM with 10% fetal bovine serum (FBS), 1 % pen-strep and 0.1 % geneticin all produced by Gibco. Cells were incubated at 37°C in a humidified incubator with 5% CO 2 . Cells were plated at 60,000 cells per well on a 96 well plate coated with rat tail collagen and incubated for 24 h.

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Abstract

La présente invention concerne des compositions et des procédés de diminution de la glycémie. Plus spécifiquement, la présente invention concerne des compositions comprenant un complexe comprenant IF et B12 conjugués à un peptide comprenant un agoniste du GLP-1. De manière avantageuse, la composition peut être administrée par voie sous-cutanée.
PCT/US2016/033426 2015-05-20 2016-05-20 Régulation glycémique améliorée à l'aide d'un facteur intrinsèque lié à un conjugué de vitamine b12 d'un agoniste du récepteur du glucagon-like peptide-1 WO2016187499A1 (fr)

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US15/575,468 US20180154007A1 (en) 2015-05-20 2016-05-20 Glycemic Control Using Intrinsic Factor Bound to A Vitamin B12 Conjugate of a Glucagon-Like Peptide-1 Receptor Agonist
US17/239,934 US11771772B2 (en) 2015-05-20 2021-04-26 Glycemic control using intrinsic factor bound to a vitamin B12 conjugate of a glucagon-like peptide-1 receptor agonist

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US201562164816P 2015-05-21 2015-05-21
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US17/239,934 Division US11771772B2 (en) 2015-05-20 2021-04-26 Glycemic control using intrinsic factor bound to a vitamin B12 conjugate of a glucagon-like peptide-1 receptor agonist

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EP3972432A4 (fr) * 2019-05-20 2023-07-05 Syracuse University Formulations pharmaceutiques et procédés d'administration d'un agent thérapeutique, de diagnostic ou d'imagerie à cd206

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EP3972432A4 (fr) * 2019-05-20 2023-07-05 Syracuse University Formulations pharmaceutiques et procédés d'administration d'un agent thérapeutique, de diagnostic ou d'imagerie à cd206

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